Transformative solutions are required to control and eliminate diseases transmitted by mosquitoes and protect human health and biosecurity. Mosquito control relies heavily on insecticides that are neurotoxic to many organisms, including humans, and ineffective against insecticide-resistant mosquitoes. The long-term goal of our research is the development of new, safer interventions to control mosquito vectors. We lead a mature research effort to develop a new class of insecticides that target G protein-coupled receptors (GPCRs) in mosquitoes. Our chemistries are selective for mosquito dopamine receptors (DARs) and operate via modes that are different to existing products. This work is significant because new mode-of-action insecticides would provide continued yet safer mosquito and disease control. Our goal on this two-year developmental project is to advance novel, complementary allosteric modulator technology to enhance the safety profile and insecticidal properties of GPCR-directed insecticides. Small molecule allosteric modulators are widely used in human medicine to improve the specificity of GPCR acting drugs and minimize adverse side-effects. Allosteric drugs can stimulate or inhibit receptor activity. These chemistries offer selectivity through binding at unique sites on the receptor and/or by causing the receptor to engage a specific cell-signaling pathway (signaling bias). Allosteric modulators of GPCRs can be detected using in vitro pharmacological assays. On this project, we will explore the potential of allosteric modulator technology at the DAR AaDOP2 from the Aedes aegypti mosquito vector of dengue and yellow fever. Our project goal is to identify and evaluate the pharmacology of mosquito DAR modulators. This objective will be accomplished via work under two Specific Aims: Specific Aim 1: Discover inhibitors that modulate activity at AaDOP2. Allosteric modulation of receptor activity can present as changes to the potency, efficacy and affinity of a chemistries at the receptor. We will develop cellular assays to identify negative allosteric modulators (NAMs) that reduce the potency, efficacy and affinity of chemistries at AaDOP2. Specific Aim 2: Discover negative allosteric modulators that selectively inhibit AaDOP2 signaling pathways. Modulation can present as the biased engagement of a receptor-mediated cell signaling pathway. Cellular assays that measure common signaling endpoints will be used to screen for biased NAMs and explore the phenomenon of signaling bias at AaDOP2. At the successful completion of the proposed exploratory studies, we will have identified and assessed the pharmacological properties of one or more NAMs active at the Ae. aegypti DAR. This will enable expanded efforts to develop unique small molecule technologies against multiple mosquito vectors of disease.